Genome-Wide Identification of GAST Family Members and Their Potential Roles in Epicotyl Dormancy in Chinese Cork Oak (Quercus variabilis)

Chinese cork oak (Quercus variabilis Blume) is a widespread tree species with high economic and ecological values. Chinese cork oak exhibits epicotyl dormancy, causing emergence heterogeneity and affecting the quality of seedling cultivation. Gibberellic acid-stimulated transcript (GAST) is a plant-specific protein family that plays a crucial regulatory role in plant growth, development, and seed germination. However, their evolution in Chinese cork oak and roles in epicotyl dormancy are still unclear. Here, a genome-wide identification of the GAST gene family was conducted in Chinese cork oak. Ten QvGAST genes were identified, and nine of them were expressed in seed. The physicochemical properties and promoter cis-acting elements of the selected Chinese cork oak GAST family genes indicated that the cis-acting elements in the GAST promoter are involved in plant development, hormone response, and stress response. Germinated seeds were subjected to gibberellins (GAs), abscisic acid (ABA), and fluridone treatments to show their response during epicotyl dormancy release. Significant changes in the expression of certain QvGAST genes were observed under different hormone treatments. QvGAST1, QvGAST2, QvGAST3, and QvGAST6 exhibited upregulation in response to gibberellin. QvGAST2 was markedly upregulated during the release of epicotyl dormancy in response to GA. These findings suggested that QvGAST2 might play an important role in epicotyl dormancy release. This study provides a basis for further analysis of the mechanisms underlying the alleviation of epicotyl dormancy in Chinese cork oak by QvGASTs genes.


Introduction
Epicotyl dormancy is a survival strategy in plants that serves as an adaptation to the environment.It provides a mechanism for delaying germination, assisting plant seeds in overcoming adverse conditions, such as freezing and drought, until the conditions become more favorable for the survival of the next generation [1].The breaking of epicotyl dormancy depends on the levels of gibberellins (GAs) and abscisic acid (ABA) in the hypocotyl of germinating seeds [2].This process is often accompanied by an increase in internal GAs and a decrease in ABA [3][4][5].Similar effects have been observed in other plants: Arabidopsis seeds treated with ABA and GAs exhibit significant differences in the content of intracellular microtubules, which is related to the mechanism of breaking epicotyl dormancy [6].Exogenous GA 3 treatment breaks the epicotyl dormancy of peony seeds [7].Effective relief of epicotyl dormancy in peony by low-temperature treatment is associated with a significant increase in endogenous GAs [2].In the process of relieving epicotyl dormancy in Polygonatum sibiricum seeds, the biosynthesis and signal transduction pathways of GA are detected [8].In the case of Paeonia emodi Wall.Ex Royle., the ABA/GA 3 ratio gradually decreases during the relief of epicotyl dormancy by cold stratification, and starch content is significantly correlated with the activities of α-amylase and β-amylase [9].Therefore, we speculated that GAs and ABA might be related in the process of relieving epicotyl dormancy.
The GAST (GA-stimulated transcripts) gene family, which is referred to as the GASA (gibberellic acid stimulated in Arabidopsis) gene family in certain plants, is widely distributed in the plant kingdom and is characterized by a C-terminal region consisting of approximately 60 amino acids, containing 12 conserved cysteine residues at specific positions, referred to as the GAST domain [10].Cysteine residues are essential for forming disulfide bonds, which play a crucial role in protein folding and interactions between proteins.This region is critical for maintaining the spatial structure and function of GAST proteins [10,11].The first identified member of the GAST family in tomatoes (Solanum lycopersicum) was called gibberellin-stimulated transcript 1 (GAST-1) [12].As different species' GAST gene family members have been identified, comprehensive insights into the functions of this gene family have been gained, including in Arabidopsis thaliana [13], Oryza sativa [14], Brassica rapa [15], Phyllostachys edulis [16], Phaseolus vulgaris [17], Pyrus pyrifolia [1], Prunus mume [18], Cucumis sativus [19], and Paeonia ostia [7].GAST genes participate in various plant growth and development processes, including seed germination, flower induction, and stem elongation, playing crucial regulatory roles [20].In Arabidopsis, most GAST genes are strongly expressed in the abscission zones of flowers and siliques [13].Peanut AhGASA1 and AhGASA18 show higher expression levels in large seeds at various stages of pod development compared to small seeds, suggesting their potential importance in pod development [21].The GAST gene family is also involved in the regulation of plant growth, development, and stress responses.Due to the conserved structure of the GAST domain, which includes 12 conserved cysteine residues, GAST proteins may play a role in plant defense responses [13].Cotton GhGAST4 and GhGAST18 are strongly induced under low-temperature conditions and effectively enhance cotton cold resistance [17].However, different GAST members in Arabidopsis may have opposing effects on development.For example, AtGASA4 exhibits a promoting effect on flower meristem, while AtGASA5 shows an inhibitory effect [22].Considering that the GAST gene family could be extensively involved in regulating the plant hormone signaling transduction network, GAST proteins also influence plant hormone responses, primarily regulated by gibberellins, and also participate in the regulation of abscisic acid and other plant hormones.Despite the significant role of GAST genes in plant growth regulation, whether they are involved in seed epicotyl dormancy release is still unknown.
Chinese cork oak (Quercus variabilis), widely distributed in East Asia [23], is a key species and resource in the formation of deciduous broad-leaved forests in the warm temperate zone and deciduous, evergreen broad-leaved forests in the northern subtropical zone [24].It holds significant ecological and economic value [25][26][27].Due to various limitations in asexual reproduction, oak species heavily rely on seeds for propagation [26].Under natural conditions, Chinese cork oak seeds exhibit epicotyl dormancy; after maturing and falling in autumn, the hypocotyl rapidly germinates and forms roots to prevent water loss, maintaining seed vitality.The epicotyl axis enters dormancy during the autumn and winter seasons and sprouts in the following spring, protecting the seedlings from freezing.In artificial seedling cultivation, the requirement for rapid and uniform germination poses a challenge, as the depth of epicotyl dormancy varies among seeds from the same batch, resulting in differences in germination times ranging from several weeks to even two months [28].This discrepancy leads to uneven seedling quality, prolongs the seedling management period, and increases labor costs [29].In the context of Chinese cork oak research, there is a lack of information regarding the GAST gene family.Overall, research on the seed dormancy of Chinese cork oak is still in its infancy and currently lacks genes of significant utility.Therefore, understanding the mechanisms underlying the relief of epicotyl dormancy in Chinese cork oak is crucial for seedling cultivation.
Here, we identified 10 GAST family genes from the genome of Chinese cork oak and speculated on the possible functions of some QvGAST genes through bioinformatics and transcriptome analysis.Additionally, to further validate the mechanisms of action of QvGAST genes in response to hormones, we conducted qRT-PCR analysis after different hormone treatments, exploring the regulatory functions of the GAST gene family in the process of relieving seed dormancy in Chinese cork oak seeds.To our knowledge, this study marks the inaugural thorough examination of GAST genes within the Chinese cork oak, providing an exhaustive assessment.Furthermore, there are few reports on the involvement of this gene family in seed dormancy.This research holds significant implications for the breeding and genetic improvement of Chinese cork oak, providing theoretical references for the seedling cultivation process of Chinese cork oak.

Plant Material
The Chinese cork oak (Quercus variabilis) seeds used in this study were collected from Chuzhou, Anhui, in 2023.

Exogenous Treatments of Different Plant Growth Regulators
Before exogenous treatments were applied, the seeds underwent 60 days of cold stratification, followed by 3 days of germination at 21 • C in an incubator.The germinated seeds (in epicotyl dormancy stage) were subjected to treatments with GA 4+7 , ABA, fluridone (FLU, an ABA biosynthesis inhibitor), and 0.02% ethanol (solvent for hormone stock solution as a control).After 12 h soak, the seeds were sowed and cultured under comfortable conditions (24 • C, with a daily light cycle of 16 h light/8 h dark, and light intensity of 5000 lx.).Subsequently, samples were taken after 12 h and 24 h of sowing and immediately frozen in liquid nitrogen for further qRT-PCR validation.

Identification of QvGAST Genes
The Chinese cork oak genome was assembled by our research group (https://figshare.com/s/67f664d5f6603982128a, accessed on 27 November 2023), and the Hidden Markov Model (HMM) file for the conserved domain of the GAST gene family was downloaded from the Pfam database (https://pfam.xfam.org/,accessed on 27 November 2023), with protein accession number PF02704.Protein sequences of Arabidopsis GASA family genes were aligned to protein sequences of Chinese cork oak.The HMMER3 3.3.2software (http://hmmer.org) was employed for searching the entire Chinese cork oak genome protein sequences, using the GAST HMM domain as the query condition.Simultaneously, amino acid sequences of Arabidopsis GASA gene family members were downloaded from TAIR11 (https://www.arabidopsis.org/,accessed on 30 November 2023).These sequences were used as query sequences, and the Chinese cork oak genome protein sequences were used as library sequences for BLAST comparison with an E-value set to 1 × 10 −5 .The combined results from both methods were used to finalize the selection of 10 QvGAST family genes.The TBtools software (https://github.com/CJ-Chen/TBtools/releases,accessed on 30 November 2023) [30], along with the Gene Location plugin, was utilized to obtain the relative chromosomal positions and gene density information of the target genes on each chromosome based on Chinese cork oak genome annotation data.Chromosome distribution was visualized accordingly.The TBtools software, specifically the Protein Parameter Calc plugin, was used for the physicochemical property analysis of the Chinese cork oak GAST gene family.The amino acid sequences of the Chinese cork oak GAST gene family were subjected to structure prediction using the SWISS-MODEL online platform (https://swissmodel.expasy.org/,accessed on 23 December 2023).Multiple results were obtained and evaluated based on GMQE (Global Model Quality Estimation) and QMEAN (Qualitative Model Energy Analysis) scores.GMQE ranges from 0 to 1, with higher values indicating better quality, while QMEAN ranges from −4 to 0, with values closer to 0 indicating better alignment with template proteins.The best model was selected based on these criteria, and the predicted protein structure was obtained.

Evolutionary Analysis of QvGAST Gene Family
To construct the evolutionary tree, the MEGA 7.0 software was employed.The fulllength protein sequences of 15 known Arabidopsis GASA gene family members, wheat TaGASR7 and TaGASR34, rice OsGSR1, and pear PpyGAST1 [1,13,31,32] and the 10 Chinese cork oak GAST gene family members were used.The parameters were set as follows: Neighbor-Joining (NJ) method, 1000 bootstrap replicates, Poisson model, and pairwise deletion.The classification of subfamilies for the Chinese cork oak GAST gene family members was performed based on the analysis method used for the published Arabidopsis GASA gene family.The obtained evolutionary tree was further enhanced for visualization using the Chiplot website (https://www.chiplot.online/#,accessed on 7 January 2024).

Gene Structure and Protein Motif Analysis of QvGASTs
For motif analysis, the MEME website (https://meme-suite.org/meme/tools/meme, accessed on 8 January 2024) was utilized.The identification of 6 motifs was set as the criterion, and the results were visualized using the TBtools software for protein-conserved motif analysis.The annotation file of the Chinese cork oak genome and the IDs of the Chinese cork oak GAST gene family members were uploaded to the TBtools software to visualize the gene structures.

Cis-Element Analysis of the Promoters of QvGASTs
The upstream 2000 bp sequences of the QvGAST gene CDS were extracted using the TBtools software.PlantCARE website (http://bioinformatics.psb.ugent.be/,accessed on 7 January 2024) was then employed to screen for cis-elements in the QvGAST promoter.Visualization of the results was performed using the TBtools software.

RNA Extraction
RNA extraction was carried out using the modified CTAB (cetyltrimethylammonium bromide) method [33].Total RNA extracted from the previously treated Chinese cork oak seed samples was used for subsequent experiments.

Gene Expression Analysis
Initially, the HiScript II Q RT SuperMix for qPCR kit was used to synthesize cDNA from 1 µg of RNA following the operating manual as previously described [34].Quantitative primers for the Chinese cork oak GAST gene family were designed using the Primer3.0 online tool (http://www.primer3plus.com,accessed on 8 December 2023).The primers are listed in Table S2.Real-time fluorescence quantitative PCR (qRT-PCR) experiments were conducted using the 7300 Real-Time PCR System (Applied Biosystems Company, Waltham, MA, USA).Data were statistically analyzed, and QvActin7 gene was used as an internal reference for normalization according to our transcriptome data and previous studies [34,35].The 2 −∆∆Ct method was employed to calculate the relative expression levels of the target genes.Statistical analyses were carried out using Sigmaplot 12.5 software based on the two-way ANOVA method, and the least significant difference test of Fisher at 0.05 significant levels was considered.All experiments were performed with four biological replicates.

Tissue-Specific Expression of QvGAST Genes
Transcriptome data from different tissues of Chinese cork oak, which were generated in our previous study [34], were analyzed to obtain the expression patterns of GAST genes in various tissues.The transcriptome data of the 10 GAST gene family members in different organs of Chinese cork oak were normalized and row-standardized using the HeatMap plugin in the TBtools software [36] for visualization.

Genome-Wide Identification of QvGAST Genes
A total of 10 QvGAST genes were identified in our recently assembled Chinese cork oak genome.Chromosomal localization analysis revealed that the 10 Chinese cork oak GAST genes were located on chromosomes 2, 3, 6, 8, and 10 (Figure 1a).Specifically, six QvGAST genes were found on chromosome 2, whereas chromosomes 3, 6, 8, and 10 each contained one QvGAST gene.The 10 QvGAST genes were renamed according to their order on the chromosomes, designated as QvGAST1 to QvGAST10.

Evolutionary Analysis of QvGAST Gene Family
To gain further insights into the evolutionary relationship among these GAST genes in Chinese cork oak and other species, we constructed an evolutionary tree using the identified 10 QvGAST protein sequences, along with 15 full-length Arabidopsis GASA protein Physicochemical property analysis (Table S1) showed that the length of the amino acid sequences encoded by Chinese cork oak GAST genes ranged from 88 aa (QvGAST1, QvGAST2, QvGAST9) to 194 aa (QvGAST5).The isoelectric point ranged from 8.42 (Qv-GAST9) to 9.59 (QvGAST3), and the molecular weight ranged from 9.62 kDa (QvGAST9) to 20.73 kDa (QvGAST5).The instability index of Chinese cork oak GAST proteins ranged from 28.90 (QvGAST8) to 91.75 (QvGAST6), the aliphatic index ranged from 45.57 (Qv-GAST9) to 80.00 (QvGAST3), and the average hydrophilicity coefficient ranged from −0.602 (QvGAST6) to 0.110 (QvGAST3).
Through the prediction of the protein structure of the Chinese cork oak GAST gene family, we found that the protein structure is mainly composed of random coils and αhelices but also includes β-folded structures (Figure 1b).It is worth noting that, compared to GAST proteins in other species, such as common beans and Chinese cabbage, the GAST proteins in Chinese cork oak also exhibit similar structural features [15,37].

Evolutionary Analysis of QvGAST Gene Family
To gain further insights into the evolutionary relationship among these GAST genes in Chinese cork oak and other species, we constructed an evolutionary tree using the identified 10 QvGAST protein sequences, along with 15 full-length Arabidopsis GASA protein sequences and other reported GAST proteins.Following the classification of the Arabidopsis GAST gene family, the Chinese cork oak GAST family members were divided into three subfamilies, namely Classes I-III (Figure 2).Class I had one member, Class II had three members, and Class III had the largest number of members, with six members.

Gene Structure and Protein Motif Analysis of QvGAST Genes
To analyze the conservation of the Chinese cork oak GAST gene family protein se quences, we visualized the gene structures.We analyzed six conserved motifs in the pro tein products.As shown in Figure 3a, the protein sequences of this family are relatively conserved, with Motif 1-4 universally present in the motifs and distributed in a highly

Gene Structure and Protein Motif Analysis of QvGAST Genes
To analyze the conservation of the Chinese cork oak GAST gene family protein sequences, we visualized the gene structures.We analyzed six conserved motifs in the protein products.As shown in Figure 3a, the protein sequences of this family are relatively conserved, with Motif 1-4 universally present in the motifs and distributed in a highly consistent arrangement.Overall, the results indicate that protein sequences in the same clustering branch share similar conserved motifs, except for QvGAST5 and QvGAST6.However, there are differences in motif distribution and quantity between individual genes.For example, QvGAST7 and QvGAST10 have Motif 5 and Motif 6 with a highly similar arrangement, suggesting they may have a specific shared function.

Cis-Acting Element Analysis of QvGAST Genes
GAST genes are widely involved in plant hormone signaling pathways and responses to abiotic stress.To understand the potential biological functions of QvGAST genes, we analyzed the cis-acting elements in the promoter regions of the Chinese cork oak GAST gene family, identifying 11 important cis-acting elements (Figure 4).These elements are mainly associated with hormone response, environmental stimuli, abiotic stress, and stress response.
The results revealed that, except for QvGAST5, nine genes have abscisic acid (ABA) response elements, and eight genes, excluding QvGAST5 and QvGAST7, have methyl jasmonate (MeJA) response elements.QvGAST2, QvGAST3, QvGAST7, QvGAST8, and QvGAST10 show GA response elements.Simultaneously, there are elements responsive to salicylic acid, auxin, zeatin, and other hormones, indicating a close association between Chinese cork oak GAST genes and hormone signal regulation.Regarding stress-related response elements, QvGAST5 contains low-temperature stress response cis-elements, suggesting its potential role in cold stress resistance.QvGAST1, QvGAST3, and QvGAST10 contain defense and stress response elements, indicating that these Chinese cork oak GAST genes may possess abilities to resist adversity and respond to stress.Additionally, light response elements are widely distributed in the GAST family, suggesting that GAST genes may be regulated by light signals.In summary, the QvGAST family participates in crucial physiological processes during Chinese cork oak growth and development.To explore the diversity of the Chinese cork oak GAST gene structures, we conducted a gene structure analysis.The results (Figure 3b) show significant differences in the numbers of exons and introns among the 10 Chinese cork oak GAST genes.In general, genes clustered together mostly have similar gene structures, indicating potential shared biological functions.All QvGAST genes have introns, and the gene structures vary, with exon numbers ranging from 2 to 4 and intron numbers from 1 to 3. Specifically, QvGAST4 has a longer UTR at the 3 ′ end, and QvGAST3 has a significantly different intron length compared to other genes.

Cis-Acting Element Analysis of QvGAST Genes
GAST genes are widely involved in plant hormone signaling pathways and responses to abiotic stress.To understand the potential biological functions of QvGAST genes, we analyzed the cis-acting elements in the promoter regions of the Chinese cork oak GAST gene family, identifying 11 important cis-acting elements (Figure 4).These elements are mainly associated with hormone response, environmental stimuli, abiotic stress, and stress response.
The results revealed that, except for QvGAST5, nine genes have abscisic acid (ABA) response elements, and eight genes, excluding QvGAST5 and QvGAST7, have methyl jasmonate (MeJA) response elements.QvGAST2, QvGAST3, QvGAST7, QvGAST8, and QvGAST10 show GA response elements.Simultaneously, there are elements responsive to salicylic acid, auxin, zeatin, and other hormones, indicating a close association between Chinese cork oak GAST genes and hormone signal regulation.Regarding stress-related response elements, QvGAST5 contains low-temperature stress response cis-elements, sug-gesting its potential role in cold stress resistance.QvGAST1, QvGAST3, and QvGAST10 contain defense and stress response elements, indicating that these Chinese cork oak GAST genes may possess abilities to resist adversity and respond to stress.Additionally, light response elements are widely distributed in the GAST family, suggesting that GAST genes may be regulated by light signals.In summary, the QvGAST family participates in crucial physiological processes during Chinese cork oak growth and development.

Tissue-Specific Expression of QvGAST Genes
To further understand the regulatory mechanisms of the Chinese cork oak GAST gene family in relieving seed dormancy and the response of Chinese cork oak seeds to GA, we utilized transcriptome data from various Chinese cork oak organ samples to analyze the gene expression levels of QvGAST family members.The transcriptome data were log-transformed, row-standardized, and used to generate a heatmap with clustering (Figure 5).Through the combination of the heatmap and TPM values from the transcriptome data, we identified transcription factors that are highly expressed in seed tissues and have relatively large TPM values in seeds.As a result, three transcription factors, namely QvGAST3, QvGAST7, and QvGAST10, were preliminarily screened.This analysis suggests that these three Chinese cork oak GAST genes may play crucial roles in regulating seed dormancy and responding to gibberellin, providing valuable insights into the molecular mechanisms underlying Chinese cork oak seed development and dormancy release.

Tissue-Specific Expression of QvGAST Genes
To further understand the regulatory mechanisms of the Chinese cork oak GAST gene family in relieving seed dormancy and the response of Chinese cork oak seeds to GA, we utilized transcriptome data from various Chinese cork oak organ samples to analyze the gene expression levels of QvGAST family members.The transcriptome data were logtransformed, row-standardized, and used to generate a heatmap with clustering (Figure 5).Through the combination of the heatmap and TPM values from the transcriptome data, we identified transcription factors that are highly expressed in seed tissues and have relatively large TPM values in seeds.As a result, three transcription factors, namely QvGAST3, QvGAST7, and QvGAST10, were preliminarily screened.This analysis suggests that these three Chinese cork oak GAST genes may play crucial roles in regulating seed dormancy and responding to gibberellin, providing valuable insights into the molecular mechanisms underlying Chinese cork oak seed development and dormancy release.

Hormonal Regulation of QvGAST Genes during Epicotyl Domancy Release
The responsive nature of GAST genes to gibberellin (GA) and abscisic acid (ABA) in other species during the process of seed dormancy release and bud dormancy release has been revealed [1,38].To further characterize the response of QvGASTs to GA and ABA during epicotyl dormancy release, the germinated seeds were treated with GA4+7, ABA, and FLU (a biosynthesis inhibitor of ABA) at different time points.We measured the expression patterns of QvGAST genes to further investigate the potential roles of hormones in Chinese cork oak seed dormancy release (Figure 6).
Two GAST genes, QvGAST5 and QvGAST9, did not show expression during this process.The results revealed that GA4+7 treatment slightly upregulated the expression of QvGAST1 and strongly induced QvGAST2, QvGAST3, and QvGAST6 within 12 h.ABA treatment led to a slight downregulation of QvGAST1, QvGAST2, and QvGAST3, a slight upregulation of QvGAST4 and QvGAST7, and a strong induction of QvGAST10 within 12 h.FLU treatment slightly upregulated QvGAST1 and QvGAST4 and significantly increased the expression of QvGAST2 and QvGAST7.The differential responses induced by GA and ABA suggest potential antagonistic roles in seed dormancy release and germination processes [33].
Importantly, the expression of these genes varied with the duration of hormone treatment.QvGAST1, QvGAST2, QvGAST3, QvGAST6, and QvGAST7 exhibited a significant upregulation in expression after 12 h of GA4+7 treatment, followed by a sharp decrease after 24 h, suggesting a feedback regulation of GA signal.Similar expression patterns were observed for QvGAST1, QvGAST2, QvGAST3, QvGAST6, QvGAST8, and QvGAST10 after FLU treatment.QvGAST10 under GA4+7 and ABA treatment and QvGAST7 under FLU treatment showed a significant upregulation after 24 h.Gene expression changes over time have been observed in studies on GAST genes in other species.Notably, the expression of QvGAST2 and QvGAST4 was significantly upregulated after 24 h (One-tailed Student's t-test, p < 0.05).Additionally, the expression of QvGAST2 was induced by gibberellin (GA), while it remained unchanged after 24 h of abscisic acid (ABA) treatment, indicating that QvGAST2 was GA responsive and ABA inhibited its expression during epicotyl dormancy release.Consequently, we speculated that QvGAST2 might be implicated in the regulatory mechanisms governing epicotyl dormancy in Chinese cork oak seeds.

Hormonal Regulation of QvGAST Genes during Epicotyl Domancy Release
The responsive nature of GAST genes to gibberellin (GA) and abscisic acid (ABA) in other species during the process of seed dormancy release and bud dormancy release has been revealed [1,38].To further characterize the response of QvGASTs to GA and ABA during epicotyl dormancy release, the germinated seeds were treated with GA 4+7 , ABA, and FLU (a biosynthesis inhibitor of ABA) at different time points.We measured the expression patterns of QvGAST genes to further investigate the potential roles of hormones in Chinese cork oak seed dormancy release (Figure 6).
Two GAST genes, QvGAST5 and QvGAST9, did not show expression during this process.The results revealed that GA 4+7 treatment slightly upregulated the expression of QvGAST1 and strongly induced QvGAST2, QvGAST3, and QvGAST6 within 12 h.ABA treatment led to a slight downregulation of QvGAST1, QvGAST2, and QvGAST3, a slight upregulation of QvGAST4 and QvGAST7, and a strong induction of QvGAST10 within 12 h.FLU treatment slightly upregulated QvGAST1 and QvGAST4 and significantly increased the expression of QvGAST2 and QvGAST7.The differential responses induced by GA and ABA suggest potential antagonistic roles in seed dormancy release and germination processes [33].
Importantly, the expression of these genes varied with the duration of hormone treatment.QvGAST1, QvGAST2, QvGAST3, QvGAST6, and QvGAST7 exhibited a significant upregulation in expression after 12 h of GA 4+7 treatment, followed by a sharp decrease after 24 h, suggesting a feedback regulation of GA signal.Similar expression patterns were observed for QvGAST1, QvGAST2, QvGAST3, QvGAST6, QvGAST8, and QvGAST10 after FLU treatment.QvGAST10 under GA 4+7 and ABA treatment and QvGAST7 under FLU treatment showed a significant upregulation after 24 h.Gene expression changes over time have been observed in studies on GAST genes in other species.Notably, the expression of QvGAST2 and QvGAST4 was significantly upregulated after 24 h (One-tailed Student's t-test, p < 0.05).Additionally, the expression of QvGAST2 was induced by gibberellin (GA), while it remained unchanged after 24 h of abscisic acid (ABA) treatment, indicating that QvGAST2 was GA responsive and ABA inhibited its expression during epicotyl dormancy release.Consequently, we speculated that QvGAST2 might be implicated in the regulatory mechanisms governing epicotyl dormancy in Chinese cork oak seeds.

Discussion
Chinese cork oak (Quercus variabilis) is a crucial forestry resource with diverse economic and ecological values.Exploring the molecular mechanisms of epicotyl dormancy release in cork oak is of significant importance in ecology and in artificial nurseries [39][40][41].Gibberellin (GA) is a class of plant hormones involved in various essential plant developmental processes, including seed dormancy release.GAST, a multi-gene family regulated by gibberellins, encodes small peptides rich in cysteine and participates in regulating plant growth and development.Through interactions with plant hormones such as gibberellin and other hormones, GAST coordinates the plant's growth regulatory network.
GAST gene family members are identified across numerous plant species, fulfilling vital functions in plant growth and developmental processes [15,21,42].Although the GAST genes have been extensively investigated in diverse plant species, their functional elucidation in perennial woody species is limited, especially during the process of seed epicotyl dormancy release.Considering the significance of the GAST gene family, we conducted a comprehensive study on the evolutionary relationships, gene structures, physicochemical properties of proteins, and promoter cis-elements of the cork oak GAST gene family.

Genome-Wide Identification and Characteristics of GAST Gene Families in Chinese Cork Oak
This study identified 10 members of the GAST gene family in the Chinese cork oak genome.This is fewer compared to the number of GAST genes identified in other species, such as Arabidopsis thaliana with 15 [13] and Prunus mume with 16 [18], suggesting a possible contraction of the gene family in Chinese cork oak.Various characteristics of the GAST protein family were examined in this study, including the number of exons and introns, isoelectric point, and molecular weight.Low molecular weight proteins consistent with other plants were found among QvGASTs.The conserved amino acid sequence at the Cterminus retains all 12 cysteine residues, which showed the characteristic of GAST proteins.QvGAST proteins were found to possess similar structures and motifs, resembling earlier findings in other plants [12,18].Further investigation revealed that most genes belonging to the same clade had similar exon-intron compositions, suggesting a correlation between the genetic makeup of the GAST domain and its evolutionary past.The distribution of GAST genes was irregular, with 10 QvGAST genes unevenly distributed across five Chinese cork oak chromosomes (Figure 1a), while no copies of QvGAST genes were found on the remaining seven chromosomes.Similar results were observed in peanut, bamboo, and plum [16,18,21].
We analyzed the evolutionary relationships among QvGAST genes.Initially, an evolutionary tree of GAST protein sequences in Chinese cork oak was constructed (Figure 2).According to the evolutionary analysis of Arabidopsis GASA genes, the identified Ah-GASA genes were classified into three subgroups (I-III).Evolutionarily, we found that the structure of QvGAST genes was closely related to their phylogeny.Two genes comprising most gene pairs were shown to have the same motif composition, indicating comparable functionality at the protein level.Furthermore, most AtGASA proteins exhibited conserved domains Motif1, Motif2, Motif3, and Motif4, which were shared among all members of the QvGAST gene family.This implies that the particular roles of the QvGAST gene family can be inferred from the conservation of genes constituting each branch within the QvGAST gene family.

Expression Patterns and Potential Functions of QvGAST Genes
Through tissue-specific expression analysis of QvGAST genes, significant differences in expression levels were observed among different genes in cupule, catkin, leaf, root, seed, and stem (Figure 5).Combined with heatmap analysis and transcriptome data in terms of TPM values, we found that QvGAST3, QvGAST7, and QvGAST10 were highly expressed in seeds; QvGAST1 and QvGAST4 showed relatively high expression levels in catkins; QvGAST5 and QvGAST8 were predominantly transcribed in roots.QvGAST2 and Qv-GAST6 exhibited relatively higher expression in leaves.Notably, the transcript of QvGAST9 was undetectable in all six examined plant organs.The distinct tissue-specific expression patterns of QvGASTs indicate that the functions of QvGAST genes have undergone tissue differentiation, suggesting functional differences in regulating the development and formation of different organs.QvGAST genes may be involved in various physiological processes of Chinese cork oak, including flowering development, leaf development, root growth, stem development, fruit maturation, and seed germination.Additionally, given that other species respond to GA and ABA during the process of seed germination [2,18,43], we investigated the expression patterns of QvGAST genes in seeds after hormonal treatments.Promoter analysis revealed the presence of numerous hormone-responsive cis-elements in the promoters of QvGASTs.A rich array of cis-elements associated with ABA responsiveness, auxin responsiveness, gibberellin responsiveness, and MeJA responsiveness was detected in the promoter regions of various QvGAST members.All QvGAST genes, except QvGAST5, possessed ABA responsiveness, whereas QvGAST2, QvGAST3, QvGAST6, Qv-GAST7, QvGAST8, and QvGAST10 are GA responsive.Expression analysis confirmed their responsiveness to exogenous GA and ABA treatments, which was validated in qRT-PCR analysis.Interestingly, QvGAST1 and QvGAST7 lacked GA-responsive elements in their promoters and exhibited induced or suppressed expression patterns after GA treatment, suggesting that these QvGAST genes might be indirectly regulated by GA-responsive factors.Apart from QvGAST1, QvGAST4, QvGAST5, and QvGAST9, the expression patterns of the remaining genes responded to exogenous GA and ABA treatments, indicating their potential roles in integrating gibberellic acid and abscisic acid signaling pathways in cork oak.Additionally, the identification of cis-elements associated with abiotic stress responses within QvGAST promoters indicates their possible participation in pertinent biological mechanisms.The GAST gene family also plays an important role in other species, such as wheat [31], where, among the 36 TaGASR genes, responsive cis-regulatory elements of five important plant hormones (ABA, SA, GA, IAA, and MeJA) were identified, along with three regulatory cis-regulatory elements for abiotic stress (such as drought, low temperature, and defense).
In recent years, research has found that the GAST family plays an important role in alleviating plant seed dormancy, bud dormancy, and other physiological activities.In many plant species, seed dormancy and germination are controlled by two main plant hormones (ABA and GA) and temperature [44,45].
Subsequently, qRT-PCR analysis was performed to examine the expression of 10 QvGAST genes at two time points under different hormonal treatments.QvGAST5 and QvGAST9 were found to be non-expressing during this process.QvGAST1, QvGAST2, QvGAST3, and QvGAST6 showed an increase in expression after GA 4+7 treatment for 12 h, indicating their regulation by gibberellins.Upon FLU treatment, QvGAST1, QvGAST2, QvGAST4, QvGAST6, and QvGAST7 showed elevated expression levels.After ABA treatment, Qv-GAST3, QvGAST4, QvGAST6, QvGAST7, and QvGAST10 exhibited increased expression.Notably, QvGAST1 and QvGAST2 cluster together in the evolutionary tree and are likely to share analogous functionalities.In contrast, the expression patterns of QvGAST7 and QvGAST10 were opposite.Although different species may exhibit variations in their evolutionary pathways, such differences do not automatically lead to significant differences in the functions of the genes or traits under consideration.In Arabidopsis, for instance, AtGASA4 promotes GA response and seed germination [46], while its homolog AtGASA5 inhibits GA signaling and seed germination under paclobutrazol (PAC, a GA biosynthesis inhibitor) treatment [47].Similarly, in rice, despite OsGASA4 and OsGASA6 having similar physiological functions, such as inducing GA and inhibiting ABA, they cluster into different subgroups [48].We observed a significant decrease in gene expression after 24 h of gibberellin treatment compared to 12 h.These could be GAST genes that promote gibberellin synthesis, and as the gibberellin content increases, it inhibits its own synthesis [1].Therefore, there may be additional mechanisms to inhibit GAST synthesis, which requires further investigation.The increase in exogenous gibberellin might lead to reduced endogenous gibberellin production, thereby resulting in a substantial decrease in gene expression.
Arabidopsis AtGASA4, AtGASA5 [46], and AtGASA6 [42], wheat TaGASR7 and TaGASR34 [31], and rice OsGSR1 [32] genes play key roles in controlling seed dormancy and germination.Pear PpyGAST1 has a promoting effect on bud dormancy release [1].Based on the evolutionary tree of the GAST family members (Figure 2), we identified that some Chinese cork oak QvGASTs are closely related to the homologs of wheat TaGASR34, TaGASR7, rice OsGASR1, and Arabidopsis AtGASA4, AtGASA6, implying that they may have similar functions.Wang found that OsGSR1 is a positive regulator of GA signaling [32].Sun reported that GA upregulates the expression of Arabidopsis AtGASA14 [49], while transcriptional regulatory factors inhibiting GA response downregulate the expression of AtGASA14.Similarly, we found that QvGAST1 and QvGAST2 on the same branch of the evolutionary tree are upregulated after GA treatment, showing increased sensitivity to GA, supporting the involvement of these QvGAST genes in GA signaling.Specifically, QvGAST2 was significantly upregulated during the process of emergence of the seeds, leading us to speculate that QvGAST2 might be involved in the regulation of epicotyl dormancy in Chinese cork oak.However, the biological role of QvGASTs in regulating epicotyl dormancy release remains largely unknown and requires further investigation in future studies.

Conclusions
This study presents the first systematic genomic analysis of the QvGAST gene family in cork oak.A total of 10 QvGAST genes were identified, enriching our understanding of the cork oak QvGAST gene family through further bioinformatics analysis.Tissue-specific expression analysis revealed high expression of QvGAST3, QvGAST7, and QvGAST10 in seeds, suggesting their potential involvement in the physiological process of seed dormancy.Furthermore, hormone treatments revealed several QvGAST genes responsive to GA and ABA.QvGAST1, QvGAST2, QvGAST3, and QvGAST6 were upregulated by gibberellin, while QvGAST3, QvGAST4, QvGAST6, QvGAST7, and QvGAST10 showed increased expression after ABA treatment.These findings suggest the close involvement of these QvGAST genes in plant hormone signaling pathways, further influencing epicotyl dormancy in Chinese cork oak.QvGAST2 was significantly upregulated during the process of releasing epicotyl dormancy in response to GA, suggesting that QvGAST2 might be involved in the regulation of epicotyl dormancy in Chinese cork oak.These discoveries provide valuable information for further investigation into the functions of these candidate genes.This study offers new insights into the evolution of the GAST gene family in cork oak and the potential molecular mechanisms underlying the integration of GA biosynthesis and ABA signaling in cork oak seed dormancy release.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants13091247/s1,Table S1: Analysis of physicochemical properties of members of GAST gene family in Chinese cork oak; Table S2

Plants 2024 , 16 Figure 1 .
Figure 1.(a) The distribution of GAST in Chinese cork oak.Color blocks from blue to red indicate a gradual increase in gene density.(b) Predicted models of protein structure of QvGAST proteins in Chinese cork oak.The blue color represents regions where the prediction results are deemed highly reliable, while the red color indicates regions with lower reliability in the prediction results.

Figure 1 .
Figure 1.(a) The distribution of GAST in Chinese cork oak.Color blocks from blue to red indicate a gradual increase in gene density.(b) Predicted models of protein structure of QvGAST proteins in Chinese cork oak.The blue color represents regions where the prediction results are deemed highly reliable, while the red color indicates regions with lower reliability in the prediction results.

16 Figure 2 .
Figure 2. Evolutionary analysis of 29 GAST family genes from Quercus variabilis, Arabidopsis thaliana Oryza sativa, Triticum aestivum, and Pyrus pyrifolia.A Neighbor-Joining evolutionary tree was con structed, substantiated by 1000 bootstrap iterations in MEGA.The GAST proteins were differenti ated into three distinct groups (Ⅰ, Ⅱ, Ⅲ), each designated with a unique color.

Figure 2 .
Figure 2. Evolutionary analysis of 29 GAST family genes from Quercus variabilis, Arabidopsis thaliana, Oryza sativa, Triticum aestivum, and Pyrus pyrifolia.A Neighbor-Joining evolutionary tree was constructed, substantiated by 1000 bootstrap iterations in MEGA.The GAST proteins were differentiated into three distinct groups (I, II, III), each designated with a unique color.

16 Figure 3 .
Figure 3. Examination of protein motifs and gene architectures for the GAST family genes discovered within the Chinese cork oak.(a) A dendrogram depicting QvGAST protein sequences, featuring conserved motifs in distinct colorations.(b) Exon-intron distribution analysis of QvGAST genes.The green boxes represent CDS, the green boxes represent UTR, and the black lines represent intron positions, respectively.

Figure 3 .
Figure 3. Examination of protein motifs and gene architectures for the GAST family genes discovered within the Chinese cork oak.(a) A dendrogram depicting QvGAST protein sequences, featuring conserved motifs in distinct colorations.(b) Exon-intron distribution analysis of QvGAST genes.The green boxes represent CDS, the green boxes represent UTR, and the black lines represent intron positions, respectively.

Plants 2024 , 16 Figure 4 .
Figure 4. Cis-acting element distribution of QvGAST gene family in Chinese cork oak.(a) Identification of cis-elements present in the promoter regions of QvGAST genes.(b) The figures and colors represent the quantity of cis-elements cataloged in the promoters of individual QvGAST genes.

Figure 4 .
Figure 4. Cis-acting element distribution of QvGAST gene family in Chinese cork oak.(a) Identification of cis-elements present in the promoter regions of QvGAST genes.(b) The figures and colors represent the quantity of cis-elements cataloged in the promoters of individual QvGAST genes.

Figure 5 .
Figure 5. Expression patterns of GAST family members in different organs of Q. variabilis.Red blocks represent high levels of TPM values, blue blocks represent low levels of TPM values, and white blocks represent the middle level of TPM values after normalization.

Figure 5 .
Figure 5. Expression patterns of GAST family members in different organs of Q. variabilis.Red blocks represent high levels of TPM values, blue blocks represent low levels of TPM values, and white blocks represent the middle level of TPM values after normalization.

Figure 6 .
Figure 6.Relative expression levels of GAST genes after GA4+7, ABA, and FLU treatments at different time points; Mock represents the control group without any hormone treatment.The horizontal axis label "12" represents 12 h after sowing, and "24" represents 24 h after sowing.The error bars indicate the standard deviation among the four biological replicates.Different letters above the column indicate significant differences among the samples (One-way ANOVA analysis with Duncan's test, p < 0.05).

Figure 6 .
Figure 6.Relative expression levels of GAST genes after GA 4+7 , ABA, and FLU treatments at different time points; Mock represents the control group without any hormone treatment.The horizontal axis label "12" represents 12 h after sowing, and "24" represents 24 h after sowing.The error bars indicate the standard deviation among the four biological replicates.Different letters above the column indicate significant differences among the samples (One-way ANOVA analysis with Duncan's test, p < 0.05).
: Oligonucleotide primer sequences used for qRT-PCR and in Chinese cork oak experiments.Author Contributions: Y.W.: conceptualization, writing-original draft, writing-review and editing, validation, visualization, software, investigation, formal analysis, data curation.Y.H.: data curation, visualization, supervision, investigation.Y.C.: investigation, data curation, visualization, formal analysis.Z.Y.: resources, software, visualization, data curation, formal analysis.P.L.: writing-review and editing, investigation.G.L.: project administration, writing-review and editing, supervision, resources.Q.Y.: conceptualization, methodology, writing-review & editing, project administration, funding acquisition, data curation, investigation, supervision, resources.All authors have read and agreed to the published version of the manuscript.